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Germline transmission of genetically modified primordial germ cells


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Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.
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© 2006 Nature Publishing Group
Germline transmission of genetically modified
primordial germ cells
Marie-Cecile van de Lavoir
, Jennifer H. Diamond
, Philip A. Leighton
, Christine Mather-Love
Babette S. Heyer
, Renee Bradshaw
, Allyn Kerchner
, Lisa T. Hooi
, Terri M. Gessaro
, Susan E. Swanberg
Mary E. Delany
& Robert J. Etches
Primordial germ cells (PGCs) are the precursors of sperm and
. In most animals, segregation of the germ line from the
somatic lineages is one of the earliest events in development
avian embryos, PGCs are first identified in an extra-embryonic
region, the germinal crescent, after approximately 18 h of incu-
bation. After 50–55 h of development, PGCs migrate to the gonad
and subsequently produce functional sperm and oocytes
. So far,
cultures of PGCs that remain restricted to the germ line have not
been reported in any species
. Here we show that chicken PGCs
can be isolated, cultured and genetically modified while main-
taining their commitment to the germ line. Furthermore, we show
that chicken PGCs can be induced in vitro to differentiate into
embryonic germ cells that contribute to somatic tissues. Retention
of the commitment of PGCs to the germ line after extended
periods in culture and after genetic modification combined with
their capacity to acquire somatic competence in vitro provides
a new model for developmental biology. The utility of the model
is enhanced by the accessibility of the avian e mbryo, which
facilitates access to the earliest stages of development and supplies
a facile route for the reintroduction of PGCs into the embryonic
vasculature. In addition, these attributes create new opportu-
nities to manipulate the genome of chickens for agricultural and
pharmaceutical applications.
Blood containing PGCs was collected from stage 14–17 (H&H;
nomenclature used in ref. 7) Barred Plymouth Rock (BPR) chicken
embryos and cultured in knockout (KO)-DMEM medium con-
ditioned on buffalo rat liver (BRL) cells (which are known to produce
leukaemia inhibitory factor), containing stem cell factor (SCF;
) and human recombinant fibroblast growth factor (FGF;
). PGCs were grown on a feeder of either Sandoz inbred
mouse-derived thioguanine-resistant and ouabain-resistant (STO)
fibroblasts or BRL cells. Twelve cell lines determined to be male by the
absence of the Xho repeat in the W chromosome
(data not shown)
and two female cell lines were derived from 114 individual embryos.
Cells in all of the lines display a round morphology, remain
unattached (Fig. 1a) and can be successfully cryopreserved using
conventional techniques. After 217 days in culture, karyotype analy-
sis of one cell line (PGC13) revealed that it was diploid with a male
ZZ sex chromosome constitution. To determine whether the cultured
PGCs maintain their germ cell characteristics we analysed the
expression of the germline-specific genes chicken vasa homologue
and deleted in azoospermia-like (DAZL). Polymerase chain
reaction with reverse transcription (RT–PCR) analysis of PGCs after
32, 143 and 197 days of culture showed expression of DAZL and CVH
(Fig. 1b), whereas at 166 days of culture, western blot analysis showed
production of the CVH protein in the cultured cells (data not
shown). Expression of CVH and an ovomucin-like protein (OLP)
Figure 1 | Characteristics of PGC cell lines in vitro. a, PGCs are not attached,
maintain a round morphology and are grown on a feeder layer of STO cells.
b, CVH and DAZL expression in PGC13 and DT40 cells. Expression of
both CVH and DAZL was observed in PGCs whereas DT40 cells did not
express either CVH or DAZL. c, TRAP assay. Repeat sequences are visible in
PGCs and the positive control cells (293), indicating the presence of
telomerase. The 36-bp band in the negative control chicken embryonic
fibroblast (CEF) lane is the internal PCR template. d, A WL is homozygous
dominant at the dominant white locus (I/I). When bred to a BPR hen (i/i) all
offspring from a WL rooster will be white (I/i). A black chick demonstrates
that cultured PGCs derived from a BPR embryo (i/i) colonized the germ line.
e, Chicken EG cells derived from PGCs in culture. EG cells are small, have
large nuclei (light grey) and pronounced nucleoli. The EG cells were derived
from PGCs isolated from black-feathered BPR embryos. f, Chimaeras
obtained after EG cell injection into stage X (EG&K) white-feathered WL
embryos. Somatic chimaerism is evident by the black feathers.
Origen Therapeutics, 1450 Rollins Road, Burlingame, California 94010, USA.
Department of Animal Science, 1 Shields Avenue, University of California, Davis, 2131D Meyer Hall,
Davis, California 95616, USA.
Vol 441|8 June 2006|doi:10.1038/nature04831
© 2006 Nature Publishing Group
that is expressed on the surface of PGCs during migration and
colonization of the gonad
were examined by fluorescence-activated
cell sorting (FACS) at 44 and 65 days for PGC102, and at 229 and 280
days for PGC13. The FACS profiles of PGCs, chicken embryonic stem
(ES) cells
and chicken embryonic germ (EG) cells revealed that the
1B3 antibody recognizes the ovomucin-like protein only on PGCs
and that the expression of CVH is likewise restricted to PGCs (Fig. 2).
By comparison, cells expressing an ES cell phenotype or an EG cell
phenotype, and DT40 cells that express a pre-B-cell phenotype
, did
not express these antigens. Between 7% and 20% of the cells in the
PGC cultures did not stain for either protein, indicating that some
cells in each culture do not express a PGC phenotype. It is possible
that the starting population was heterogeneous; however, a small part
of the PGC population regularly attaches to the feeder layer and
differentiates. This differentiation is also seen in clonally derived cell
lines, indicating that the non-stained cells are differentiated PGCs. A
telomeric repeat amplification protocol (TRAP) assay performed on
PGCs that were in culture for 196 days demonstrated the presence of
telomerase, which is a key characteristic of immortal cell lines (Fig. 1c).
The presence of telomerase is consistent with the long-term growth of
the PGCs in culture.
To demonstrate that PGCs maintain their restriction to the germ
line, cells from eight male cell lines that were in culture for a
minimum of 35 days and a maximum of 110 days were injected
into stage 13–15 (H&H) White Leghorn (WL) embryos, which are
post-gastrulation embryos with 19–27 somites and a functional
vasculature. Twenty-four male chimaeric chicks were reared to sexual
maturity. Each of these chimaeras displayed the white-feathered
phenotype of the WL breed (Fig. 1d). All of the male chimaeras
transmitted the PGC phenotype (recognizable by black feathers) to
the next generation, demonstrating that the cells retained their ability
to colonize the germ line (Table 1 and Fig. 1d). The rate of germline
transmission of these birds ranged from ,1% to 86%, which is
similar to the rates of germline transmission obtained in single-sex
chimaeras after the transfer of freshly isolated PGCs
. Female PGCs
in culture for 47 and 66 days were transmitted to the next generation
by female chimaeras at frequencies up to 69% (Table 1), although
these cell lines could not be maintained beyond 109 and 77 days,
respectively. The variability of germline transmission among hens
and roosters injected with aliquots of the same pool of cells may be
due to variation in the number of injected PGCs that colonize the
germ line and differential expansion of each of the cells within the
gonad. Germline transmission of male PGCs has not been observed
in 1,625 offspring of 14 female putative chimaeras, and female PGCs
did not colonize the germ line of three male putative chimaeras that
produced 2,739 offspring when mated to BPR hens. Similarly low
rates of germline transmission were observed in mixed-sex chimaeras
produced with freshly isolated PGCs
, indicating that the develop-
ment of germ cells is impaired in mixed-sex chimaeras. To evaluate
the reproductive capacity of offspring derived from cultured PGCs,
male and female progeny were mated together. Fertility ranged from
53% to 85% and the hatch rate of fertile eggs ranged from 79% to
96%, indicating that the reproductive capacity of offspring derived
from PGCs in culture for 40 days is normal.
Conventional electroporation protocols that we use routinely
to introduce genetic modifications into chicken ES cells failed to
yield genetically modified PGCs. To obviate potential silencing of
transgenes, two copies of the HS4 insulator sequence from the
-globin locus
were inserted 5
and 3
of the transgenes
and genetically modified clones were isolated. PGCs carrying an
-actin-puro-HS4 transgene (see Methods) were
injected after 134 days in culture into stage 13–15 (H&H) embryos and
eight roosters were bred to BPR hens to evaluate germline trans-
mission. Seven of the eight roosters transmitted the PGC-derived
genotype through the germ line at frequencies varying from 1% to
92%. Eighty of the 163 black-feathered offspring (49%) exhibited
green fluorescence (Fig. 3b), indicating mendelian segregation of the
transgene among PGC-derived offspring. Southern blot analysis
confirmed that chicks expressing GFP were derived from the trans-
genic PGC line (Fig. 3a) and all of the chicks carrying a copy of the
transgene expressed GFP (data not shown). We have also obtained
transfected male cell lines carrying an HS4-ERNI-neo or an HS4-
actin-puro transgene and have hatched live chicks carrying an HS4-
-actin-neo transgene from PGCs in culture for 267 days and an
HS4-CAG-EGFP-CAG-puro transgene from PGCs in culture for
238 days.
A small proportion of PGCs attaches spontaneously to the feeder
layer and assumes the morphology of ES cells, which is shown in
Figure 2 | FACS analysis of DT40 cells, ES cells, EG cells and PGCs,
stained with antibodies against CVH and OLP. The DT40, ES and EG cells
were negative for both markers, whereas a large majority of PGCs stained for
both CVH and OLP.
Table 1 | Germline transmission of primordial germ cells injected into the vasculature of stage 14–15 (H&H) embryos
Cell line Sex Time in culture (days) No. of cells injected No. of putative chimaeras tested Germline transmission* (%)
PGC13 Male 40 1,200 3 0.1,1.5,17
PGC13 Male 110 2,500–3,000 5 1,1,1.5,3,84
PGC21 Male 44 1,500 3 10,16,21
PGC34 Male 47 3,000 3 42,74,80
PGC35 Male 35 3,000 7 15,23,47,61,80,85,86
PGC51 Male 47 3,000 1 11
PGC54 Male 47 3,000 4 0.5,2,20,24
PGC80 Male 29 3,000 1 55
PGC84 Male 50 3,000 1 70
PGC56 Female 66 3,000 5 1,3,6,52,69
PGC85 Female 47 3,000 10 0,0,1,1,1,4,5,10,11,12
*Each value represents the rate of germline transmission of one chimaera.
NATURE|Vol 441|8 June 2006 LETTERS
© 2006 Nature Publishing Group
ref. 11. By removing FGF, SCF and chicken serum these cells can be
expanded and are called EG cells (Fig. 1e). Without these compo-
nents and with an increase in the percentage of conditioned medium,
the culture conditions are the same as those used for long-term
culture of ES cells derived from blastodermal cells
. Chicken EG cells
are observed in both newly derived and clonally derived transgenic
PGC lines. Southern blot analysis of EG cells derived from a clonal
GFP-positive PGC line (Fig. 3a) indicates that EG cells originate from
the PGCs and are not derived from a contaminating population of
cells in blood samples from stage 14–17 (H&H) embryos from which
the cultures originated. When EG cells were injected into stage X
(EG&K; nomenclature used in ref. 15) embryos, which are the
functional equivalents of mammalian blastocysts, they contributed
extensively to various somatic tissues (Fig. 1f and Supplementary
Fig. 1). Of the 140 WL embryos injected with EG cells, 30 survived
until at least embryonic day (E)14, and 20 embryos (67%) exhibited
the black feathering of the BPR breed that was used to derive the EG
cells. Four of these chimaeras with extensive feather pigmentation
have been bred to evaluate contributions to the germ line. One black
chick was observed among 4,490 offspring, indicating that some
capability to colonize the germ line is retained within the culture. In
general, however, the ability of EG cells to colonize the somatic tissues
and their inability to colonize the germ line is similar to that of
chicken ES cells
To determine whether cultured PGCs contribute to somatic
tissues, tissues from chicks injected with GFP-positive PGCs were
analysed by histology. With the exception of four green cells in a brain
sample, cells expressing GFP were not present in other tissues,
demonstrating that cells in a PGC culture remain restricted to the
germ line. When PGC13 cells, after 209 days of culture, were injected
into the subgerminal cavity of 111 stage X (EG&K) embryos, 20
embryos survived to E14 but black feathers were not observed,
indicating that PGCs do not contribute to somatic tissues, even
when injected at very early stages of development. However, three out
of four roosters injected with PGCs at stage X (EG&K) transmitted
them through the germ line at frequencies of 0.15%, 0.2% and 0.45%,
indicating that they can colonize the germ line of early stage embryos.
In contrast, chicken ES cells contribute substantially to somatic
tissues but not to the germ line
We describe a novel system for the production of transgenic
chickens using PGCs. To our knowledge this is the first description
in any species of PGCs that can be grown indefinitely in vitro and be
genetically manipulated while retaining their commitment to the
germ line. Combined with the extensive anatomical database
describing development in the chick embryo and the ease of access
to the earliest stages of development
, the ability to make genetic
changes to chickens will provide a unique resource to address
important issues in developmental biology. In addition, the ability
to use cultured PGCs to make changes to the germ line of chickens has
enormous implications for industrial
and agricultural applications
of avian transgenic technology.
Derivation, culture and cryopreservation of PGCs. One to five microlitres of
blood was taken from the vasculature of stage 14–17 (H&H) embryos and
deposited in single wells of a 96- or 48-well plate seeded with either mitotically
inactivated STO cells (3 £ 10
cells cm
) or BRL cells (10
cells cm
). After
1–2 weeks, red blood cells had died and PGCs became visible. Throughout
derivation and culture, the cells were grown in KO-DMEM (Invitrogen) that was
conditioned with BRL cells
. The medium was supplemented with 7.5% fetal
calf serum (FCS), 2.5% chicken serum, 2 mM glutamine, 1 mM pyruvate, 1£
nucleosides, 1£ non-essential amino acids and 0.1 mM
SCF and 4 ng ml
human recombinant FGF. For passage, the cells and
medium were removed into a centrifuge tube, pelleted by centrifugation at 300 g
for 5 min, resuspended and seeded at a concentration of 25,000 cells cm
. For
cryopreservation, the cells were resuspended in CO
-independent medium
containing 10% FCS, 1.0% penicillin/streptomycin and 10% DMSO. The vials
were frozen at 280 8C and transferred to LN
after 24 h.
RNA isolation and RT–PCR. A 750-base-pair (bp) fragment from the CVH
transcript, a 536-bp fragment from the DAZL transcript and a 597-bp fragment
from the chicken
-actin transcript were amplified by RT–PCR (Supplementary
Table 1).
FACS analysis of PGCs, EG cells and ES cells. Cells were washed in PBS/2% FBS
Figure 3 |
-Actin-EGFP is incorporated into the PGC genome and
expressed in offspring.
a, Southern blot analysis showing that a clonally
derived, transfected PGC line can contribute to the germ line in chimaeric
chickens and differentiate into EG cells. Top panel: samples of genomic DNA
from PGCs transfected with the HS4-
-actin-puro construct,
three embryos derived from a chimaeric rooster made with the transfected
PGCs, and EG cells derived from the transfected PGCs were digested with
restriction enzymes for detecting internal (KpnI) and junction fragments
(NcoI, AflII) of the transgene insertion. The digested DNA was separated on
a 0.7% agarose gel, blotted to nylon membrane, and probed with
radiolabelled EGFP sequences. The sizes of the hybridizing fragments in
kilobases (left axis) were identical in the PGCs, EG cells and two embryos
that showed green fluorescence (GFP
embryos). A third, non-fluorescing
embryo (WT embryo) showed no hybridization. Bottom panel: a schematic
of the construct is shown, with the locations of the restriction sites indicated,
and the expected restriction fragment sizes shown below. b, The offspring of
a chimaeric rooster transgenic for the HS4-
transgene at embryonic stage 34 (H&H). GFP is expressed ubiquitously
throughout the embryo. The image was captured on a Leica MZ16FA
microscope fitted with a Leica DFC300FX camera and a PlanAPO 0.6£ lens
that were provided by Leica.
LETTERS NATURE|Vol 441|8 June 2006
© 2006 Nature Publishing Group
and fixed in 4% paraformaldehyde for 5 min. Cell aliquots to be stained for CVH
were permeabilized with 0.1% Triton X-100 for 1–2 min. Primary antibody was
added for 20min, cells were washed twice and incubated with secondary antibody
(Alexa 488 anti-rabbit IgG for CVH and control and Alexa 488 anti-rabbit IgM for
1B3) for 15 min. Control cells were stained only with secondary antibody.
Telomerase detection. Primordial germ cells that were in culture for 196 days
were pelleted and washed with PBS before being frozen at 280 8C until analysis.
Cell extracts were prepared according to the manufacturer’s directions using the
TRAPeze telomerase detection kit (Serologicals Corporation)
. The positive
control was the transformed human kidney cell line 293 and the negative control
contained lysis buffer and an internal PCR control template.
Karyotype analysis. PGCs were incubated in 0.1
colcemid (Karyomax,
Invitrogen) for 2 h. The cells were pelleted and resuspended in 0.56% KCl
solution. After 25 min, the cells were centrifuged for 6 min at 200 g and the pellet
was fixed in 3:1 methanol:glacial acetic acid. For metaphase analysis the cells
were dropped on slides, stained with Giemsa and analysed for the four pairs of
macro-chromosomes (GGA1– GGA4) and the sex chromosomes.
Transfection of PGCs. A total of 5 £ 10
PGCs were resuspended in 400
electroporation buffer (Speciality Media) to which 20
g of linearized DNA was
added. One exponential decay pulse (200 V, with 900–1,100
F) or eight square
wave pulses (250–350 V, 100
s) were given. After transfection the cells were
grown for several days before neomycin (300
g ml) or puromycin (0.5
g ml)
was added. In most cases, single colonies were derived by plating the cells at low
concentration in 48-well plates after transfection.
Transgene construction. Transgenes were flanked by the insulator element from
the chicken
-globin locus. The 250-bp core sequence of hypersensitive site 4
(HS4) was amplified from chicken genomic DNA by PCR (Supplementary Table
1) and a tandem duplication of the HS4 site was made by joining two copies of
the PCR product, in the same orientation, resulting in 2£ HS4. The 2£ HS4
insulator was then inserted at both the 5
and 3
ends of the following constructs:
-actin-neo (gifts from J.-M. Buerstedde),
puro, ERNI-neo
Production of chimaeras using PGCs. PGCs were injected using a 37-
diameter needle into the anterior portion of the sinus terminales of a stage 13–15
(H&H) embryo. The injected embryos were transferred to a second surrogate
shell for incubation until hatching
Derivation of EG cells and production of chimaeras. When an EG cell
phenotype became visible, the growth factors and chicken serum were removed
from the medium. Chicken EG cells were grown in 80% BRL conditioned
medium on irradiated STO feeders plated at 10
cells cm
confluency the cells were incubated in Ca/Mg free PBS to obtain small clumps
and passaged 1:2 or 1:3. For the production of chimaeras the cells were
trypsinized into a single cell suspension and resuspended at 5,000 cells
One microlitre was injected into the subgerminal cavity of an irradiated stage X
(EG&K) embryo. After injection the embryo was incubated for 3 days before
being transferred to a second surrogate shell and incubated until hatching
Received 30 September 2005; accepted 12 April 2006.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank C. Gitter for technical assistance with the
karyotype; A. Pradas-Monne for help in the laboratory; W. Halfter for providing
the 1B3 antibody; Leica for the provision of optical equipment to photograph the
GFP-positive embryo; and J.-M. Buerstedde for supplying
-actin-neo and
-actin-puro. This work was supported by the Small Business Innovation
Research Programs of the USDA and the NIH to Origen Therapeutics and a
USDA grant to M.E.D.
Author Contributions M.C.L. developed the cell culture system with the
assistance of J.H.D., P.A.L. and R.B.; C.M.-L. and J.H.D. performed the
embryological manipulations; P.A.L. executed the molecular biology in
collaboration with B.S.H. and L.T.H.; A.K. provided animal care; T.M.G., S.E.S.
and M.E.D. conducted the telomerase assay and karyotyping; M.C.L. and R.J.E.
coordinated the contributions of authors and wrote the paper. All authors
discussed the results and commented on the manuscript.
Author Information Reprints and permissions information is available at The authors declare competing
financial interests: details accompany the paper at
Correspondence and requests for materials should be addressed to R.J.E.
NATURE|Vol 441|8 June 2006 LETTERS
... The first highly efficient generation of genetically engineered birds (GEBs) was involved in the production of "GFP-expressing" chicks using a viral vector [10]. The landmark technology in the field was the feeder-dependent long-term culture of chicken primordial germ cells (cPGCs) and generation of GEBs using the in vitro engineered cPGCs [11]. From a technological perspective, the production of the FDA-approved "Kanuma" was a great achievement that paved the way for the use of GEBs for the production of glycoproteins [2]. ...
... Although the efficiency of viral-mediated transgenesis was reasonable in some studies, related setbacks such as safety concerns (e.g., RVs prefer to be integrated into the vicinity of promoters, while transcription units are targeted by LVs, reviewed in [114]; Figure 4), being labor-intensive, reduced survivability, high rate of random integration, and progressive silencing shifted the perspective of researchers into the use of "non-viral expression plasmid." Also, with the improvements in the ability to isolate and culture chicken stem/germ cells in long term [11,115], avian in vivo genome engineering (embryo-mediated transgenesis) changed to in vitro genome engineering (cell-mediated transgenesis) (Table 1 and Figure 2). ...
... Considering the inability of cESCs and chicken embryonic germ cells to contribute to the germline, long-term in vitro cultured cPGCs were the next candidates for random integration by plasmids [11]. Plasmids with four types of promoters (CMVp, CBAp, ERNI promoter; ERNIp, CAGp) were electroporated into the cPGCs. ...
Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-CRISPR and CRISPR era in the field of avian genome engineering.
... Alternatively, completely removing embryos from their natural eggshells to various types of recently developed artificial vessels for further observation of developmental changes and genetic manipulation is called ex ovo studies. To provide in-depth studies into avian embryogenesis, parts of embryos were taken to derive cell lines in in vitro culture including embryonic stem cells (ESCs) and primordial germ cells (PGCs), and become an essential tool to understand pluripotency network regulating early development (van de Lavoir et al., 2006;Choi et al., 2010;Aubel and Pain, 2013;Whyte et al., 2015;Altgilbers et al., 2021). FIGURE 1 | Perspective view of avian embryonic culture with ex ovo-in ovo cultivation and in vitro cell culture. ...
... The discovery of chick ESCs and blastodermal cell culture emphasizes the conserved network regulating pluripotency required at least leukemia inhibitory factor (LIF) (Etches et al., 1997;Pain et al., 1996), a cytokine used to culture naïve mouse ESCs and iPSCs (Niwa et al., 1998;Takahashi and Yamanaka, 2006). In addition, basic fibroblast growth factor (bFGF), a cytokine used to cultivate primed human ESCs/iPSCs (Thomson et al., 1998;Takahashi et al., 2007), is required in several avian ESCs/iPSCs studies (Pain et al., 1996;van de Lavoir et al., 2006;Whyte et al., 2015;Choi et al., 2016;Katayama et al., 2018). Thus, this suggests that pluripotent stem cells from avian species exhibit some bias in a naïve-primed direction which could depend on other culture supplements. ...
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The avian embryos growing outside the natural eggshell (ex ovo) were observed since the early 19th century, and since then chick embryonic structures have revealed reaching an in-depth view of external and internal anatomy, enabling us to understand conserved vertebrate development. However, the internal environment within an eggshell (in ovo) would still be the ideal place to perform various experiments to understand the nature of avian development and to apply other biotechnology techniques. With the advent of genetic manipulation and cell culture techniques, avian embryonic parts were dissected for explant culture to eventually generate expandable cell lines (in vitro cell culture). The expansion of embryonic cells allowed us to unravel the transcriptional network for understanding pluripotency and differentiation mechanism in the embryos and in combination with stem cell technology facilitated the applications of avian culture to the next levels in transgenesis and wildlife conservation. In this review, we provide a panoramic view of the relationship among different cultivation platforms from in ovo studies to ex ovo as well as in vitro culture of cell lines with recent advances in the stem cell fields.
... Для получения генетически модифицированных особей используют, как правило, комплекс методов и методических подходов, учитывая объект исследований, выбор клеток-мишеней для введения рекомбинантной ДНК и способ генетической трансформации клеток-мишеней. Можно выделить три основные стратегии создания генетически модифицированной птицы: введение генетических конструкций непосредственно в эмбрион (27,28) или в органы и ткани взрослых особей (29,30); трансфекция клеток-мишеней в культуре in vitro и их последующая трансплантация в эмбрион или органы-мишени (31,32); трансформация спермиев in vitro и осеменение самок трансформированной спермой (33). ...
... In the chicken, however, this approach is hampered by the laboriousness and low efficacy of the procedures leading to the genetic knock-out or gene editing. This technology mostly relies on in vitro manipulation of PGCs, which must be reintroduced into embryos and matured to functional sperm [37]. Up to now, only a few examples of genetic knockout have been reported [38,39]. ...
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The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva −/− chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/− siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.
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As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.
Primordial germ cells (PGCs) are the undifferentiated progenitors of the gametes. Unlike the poor maintenance of cultured mammalian PGCs, the avian PGCs can be expanded in vitro indefinitely while preserving pluripotency and germline competence. In mammals, the Oct4 is the master transcription factor that ensures the stemness of pluripotent cells such as PGCs, but the specific function of Oct4 in chicken PGCs remains unclear. As expected, the loss of Oct4 in chicken PGCs reduced the expression of key pluripotency factors and promoted the genes involved in endoderm and ectoderm differentiation. Furthermore, the global active chromatin was reduced as shown by the depletion of the H3K27ac upon Oct4 suppression. Interestingly, the de-activated chromatin caused the down-regulation of adjacent genes which are mostly known regulators of cell junction, chemotaxis and cell migration. Consequently, the Oct4-deficient PGCs show impaired cell migration and could not colonize the gonads when re-introduced into the bloodstream of the embryo. We propose that, in addition to maintaining pluripotency, the Oct4 mediated chromatin activation is dictating chicken PGC migration.
Even though the chicken genome was the first livestock genome to be completely sequenced, technologies to manipulate the avian genome were lagging behind other model and agricultural animal species. This is mainly due to a complex reproductive system and the lack of embryonic stem cell cultures contributing to the germline like in mice. With the availability of lentiviral gene transfer technologies, it became possible to generate transgenic chickens on a more routine basis but induction of specific knockouts and introduction of precise edits were still not possible. The possibility to cultivate and genetically manipulate primordial germ cells (PGCs) changed the field and allows now complex modifications of the chicken genome and the subsequent generation of genetically modified chickens. At the same time, the direct in ovo manipulation of migrating PGCs became possible. The availability of viral gene transfers, PGC culture, and different ways to directly manipulate the chicken genome in ovo paved the way to generate any given modification of the chicken genome.
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The aim of the work was to develop a methodology for the creation of transgenic chimeras of ducks by using donor blastodermal cells after transfection with DNA vector and Lipofectamine 2000® (Invitrogen, USA). The CRISPR/Cas9 system with homology directed repair (HDR) was used to edit the target site of the duck genome. Materials and research methods. Transgenic duck chimeras were created using donor blastodermal cells after transfection with plasmid DNA and Lipofectamine 2000. To edit the target region of the duck genome, we used the CRISPR / cas9 system with HDR. The EGFP reporter gene was used as the transgene. Сonclusions. Среди выживших фертильных животных было 13/20 животных G0 (65 %): 10/12 (83,3 %) Of the 200 eggs, in which the transfected blastodermal cells were introduced, 20 offspring were obtained, including 8 males and 12 females. Thus, the survival of embryos was 10 %. Among the surviving fertile animals, 13/20 were animals G0 (65 %): 10/12 (83.3 %) females and 3/8 (37.5 %) males. The procedure of obtaining chimeras has a stronger effect on the survival and fertility of male chimeras. From 13 of 20 birds G0, we received a total of 197 offspring (including 117 (59.4 %) daughters and 80 (40.6 %) sons), 59 of which were EGFP-positive (30.3 %), including 10 males (16.9 %) and 49 females (83.1 %). The technique used by us can be successfully applied in further researches and for creation of a transgenic duck
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We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the β-actin promoter, a 69% Subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 μg/ml) effectively select for transfectants containing a high vector copy number (> 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.
The germ cell lineage in the mouse is not predetermined, but is established during gastrulation, in response to signaling molecules acting on a subset of epiblast cells that move through the primitive streak together with extra-embryonic mesoderm precursors. The germ cell lineage terminates in the differentiation of the gametes (eggs and spermatozoa). In mammals the lineage arises in the extraembryonic mesoderm at the posterior end of the primitive streak. During this period, they proliferate at a steady rate and are known as primordial germ cells (PGCs). PGCs do not at any stage constitute a stem cell population: each of the cell divisions that they undergo (9 or 10 in the mouse, more in the human) moves them further along their developmental trajectory. After migration to the site of the future gonads, germ cell sex determination is achieved, with germ cell phenotype in male and female embryos diverging. Site-specific DNA methylation of imprinted genes is erased in germ cells at about the time of entry into the future gonads, and new imprints are established later. Germ cells respond to certain growth factors by proliferating indefinitely. These immortalized embryonic germ cell lines are chromosomally stable and pluripotent, closely resembling the embryonic stem cell lines derived from blastocyst-stage embryos.
Since the first reports of isolation and characterization of pluripotent cell lines derived from mouse blastocysts, avian biologists have watched the application of murine embryonic stem (ES) cells in experimental biology with some envy, particularly when ES cells have been used to generate targeted changes to the mouse genome. Hence, the main impetus for the isolation and culture of avian ES cells has been the hope that such cells could be used to generate transgenic birds, with specific modifications to the avian genome. Given the current limitations associated with the production of transgenic chickens through microinjection of DNA or the use of retroviral vectors, the culture of pluripotent avian ES cells would be a valuable tool for a variety of applications in the laboratory and industrial arena. Both strategies are based on similar approaches used to isolate mammalian ES cells from early embryos but with specific modifications for the distinctiveness of the avian embryo. Many approaches toward the culture of avian ES cells have taken a cue from procedures for mouse with varying degrees of success. However, given the success of mammalian ES cells as a tool for experimental biology, it will take time to develop transgenic avian using cultured cells from either source.
1. Embryos of the domestic fowl (72 h old) have been explanted into shell‐less cultures or ‘surrogate’ eggshells, in order to investigate the possibility of rearing these embryos to hatching.2. Rocking embryo cultures during the first half of incubation enhanced embryo growth.3. Embryos explanted into ‘surrogate’ eggshells of either other individuals or other species have been successfully ‘hatched’.4. A normal chorioallantois is formed in these surrogate eggshells. This enables a functional albumen sac to form and eggshell resorption to be achieved.5. Embryos grown in ‘surrogate’ eggshells are slightly smaller than controls but otherwise normal.6. The technique provides opportunities for genetic engineering experiments.
Nucleotide sequences of three independently cloned repeating units of the W chromosome-specific repettive DNA sequences (XhoI family) of the chicken were determined. All three units are 717 bp long with XhoI sites at both ends. There are only 21 sites out of 717 bases where a single base change occurs in one of the three clones. Each of these repeating units consists of 34 tandem repeats of about 21 bp. Sequences of some members of these internal repeats are not well conserved, but the majority of the repeats are characterized by the presence of (A)3–5 and (T)3–5 clusters separated by 6–7 relatively G+C-rich base pairs. One striking feature of the cloned 717 bp repeating units is that they migrate unusually slowly on electrophoresis in polyacrylamide gels. The same feature is also shown by a genomic population of the 0.7 kb repeating units recovered from XhoI digests of the genomic DNA of the female chicken. This anomalous behavior is attributed to the occurrence of DNA curvatures because of the above sequence characteristics and partial recovery of the electrophoretic mobility in the presence of distamycin A. Another feature of the 717 bp repeating unit is the presence of 438 and 159 nucleotide-long open reading frames (ORFs) at each end of the unit. A possible function of the XhoI family sequences in the heterochromatization of the W chromosome and the significance of the ORFs are discussed.